Use of Windowed Mass Spectrometry Data for Retention Time Determination or Confirmation

ABSTRACT

A scan of a separating sample is received by a mass spectrometer at each interval of a plurality of intervals. The spectrometer performs at each interval one or more mass spectrometry scans. The scans have one or more sequential mass window widths in order to span an entire mass range at each interval and produce a collection of spectra for the entire mass range for the plurality of intervals. One or more peaks at one or more different intervals in the collection of spectra are identified for a fragment ion. A mass spectrum of the entire mass range is retrieved for each interval of each peak. Values for one or more ion characteristics of a mass-to-charge ratio peak in the mass spectrum corresponding to each peak are compared to one or more known values for the fragment ion. Each peak is scored based on the comparison.

CROSS REFERENCE TO RELATED APPLICATION

This application claims the benefit of U.S. Provisional PatentApplication Ser. No. 61/581,423, filed Dec. 29, 2011, the content ofwhich is incorporated by reference herein in its entirety.

INTRODUCTION

Mass spectrometers are often coupled with chromatography or otherseparation systems in order to identify and characterize elutingcompounds of interest from a sample. In such a coupled system, theeluting solvent is ionized and a series of mass spectra are obtainedfrom the eluting solvent at specified time intervals. These timeintervals range from, for example, 1 second to 100 minutes or greater.The series of mass spectra form a chromatogram.

Peaks found in the chromatogram are used to identify or characterize acompound of interest in the sample. In complex mixtures, however,interference with other peaks having the same mass-to-charge ratio (m/z)can make it difficult to determine a peak representing a compound ofinterest. In some cases, no information is available regarding theexpected retention time of the compound of interest. In other cases, anapproximate retention time of the compound of interest may be known.However, even in this latter case, the exact peak of the compound ofinterest can be ambiguous if the sample is complex or if there is morethan a small amount of retention time variation between samples. As aresult, it is often difficult to identify or characterize the compoundof interest in these cases.

BRIEF DESCRIPTION OF THE DRAWINGS

The skilled artisan will understand that the drawings, described below,are for illustration purposes only. The drawings are not intended tolimit the scope of the present teachings in any way.

FIG. 1 is a block diagram that illustrates a computer system, inaccordance with various embodiments.

FIG. 2 is a schematic diagram showing a system for scoring peaks of aknown compound of interest from a collection of spectra, in accordancewith various embodiments.

FIG. 3 is an exemplary flowchart showing a method for scoring peaks of aknown compound of interest from a collection of spectra, in accordancewith various embodiments.

FIG. 4 is a schematic diagram of a system that includes one or moredistinct software modules that perform a method for scoring peaks of aknown compound of interest from a collection of spectra, in accordancewith various embodiments.

Before one or more embodiments of the present teachings are described indetail, one skilled in the art will appreciate that the presentteachings are not limited in their application to the details ofconstruction, the arrangements of components, and the arrangement ofsteps set forth in the following detailed description or illustrated inthe drawings. Also, it is to be understood that the phraseology andterminology used herein is for the purpose of description and should notbe regarded as limiting.

DESCRIPTION OF VARIOUS EMBODIMENTS Computer-Implemented System

FIG. 1 is a block diagram that illustrates a computer system 100, uponwhich embodiments of the present teachings may be implemented. Computersystem 100 includes a bus 102 or other communication mechanism forcommunicating information, and a processor 104 coupled with bus 102 forprocessing information. Computer system 100 also includes a memory 106,which can be a random access memory (RAM) or other dynamic storagedevice, coupled to bus 102 for storing instructions to be executed byprocessor 104. Memory 106 also may be used for storing temporaryvariables or other intermediate information during execution ofinstructions to be executed by processor 104. Computer system 100further includes a read only memory (ROM) 108 or other static storagedevice coupled to bus 102 for storing static information andinstructions for processor 104. A storage device 110, such as a magneticdisk or optical disk, is provided and coupled to bus 102 for storinginformation and instructions.

Computer system 100 may be coupled via bus 102 to a display 112, such asa cathode ray tube (CRT) or liquid crystal display (LCD), for displayinginformation to a computer user. An input device 114, includingalphanumeric and other keys, is coupled to bus 102 for communicatinginformation and command selections to processor 104. Another type ofuser input device is cursor control 116, such as a mouse, a trackball orcursor direction keys for communicating direction information andcommand selections to processor 104 and for controlling cursor movementon display 112. This input device typically has two degrees of freedomin two axes, a first axis (i.e., x) and a second axis (i.e., y), thatallows the device to specify positions in a plane.

A computer system 100 can perform the present teachings. Consistent withcertain implementations of the present teachings, results are providedby computer system 100 in response to processor 104 executing one ormore sequences of one or more instructions contained in memory 106. Suchinstructions may be read into memory 106 from another computer-readablemedium, such as storage device 110. Execution of the sequences ofinstructions contained in memory 106 causes processor 104 to perform theprocess described herein. Alternatively hard-wired circuitry may be usedin place of or in combination with software instructions to implementthe present teachings. Thus implementations of the present teachings arenot limited to any specific combination of hardware circuitry andsoftware.

The term “computer-readable medium” as used herein refers to any mediathat participates in providing instructions to processor 104 forexecution. Such a medium may take many forms, including but not limitedto, non-volatile media, volatile media, and transmission media.Non-volatile media includes, for example, optical or magnetic disks,such as storage device 110. Volatile media includes dynamic memory, suchas memory 106. Transmission media includes coaxial cables, copper wire,and fiber optics, including the wires that comprise bus 102.

Common forms of computer-readable media include, for example, a floppydisk, a flexible disk, hard disk, magnetic tape, or any other magneticmedium, a CD-ROM, digital video disc (DVD), a Blu-ray Disc, any otheroptical medium, a thumb drive, a memory card, a RAM, PROM, and EPROM, aFLASH-EPROM, any other memory chip or cartridge, or any other tangiblemedium from which a computer can read.

Various forms of computer readable media may be involved in carrying oneor more sequences of one or more instructions to processor 104 forexecution. For example, the instructions may initially be carried on themagnetic disk of a remote computer. The remote computer can load theinstructions into its dynamic memory and send the instructions over atelephone line using a modem. A modem local to computer system 100 canreceive the data on the telephone line and use an infra-red transmitterto convert the data to an infra-red signal. An infra-red detectorcoupled to bus 102 can receive the data carried in the infra-red signaland place the data on bus 102. Bus 102 carries the data to memory 106,from which processor 104 retrieves and executes the instructions. Theinstructions received by memory 106 may optionally be stored on storagedevice 110 either before or after execution by processor 104.

In accordance with various embodiments, instructions configured to beexecuted by a processor to perform a method are stored on acomputer-readable medium. The computer-readable medium can be a devicethat stores digital information. For example, a computer-readable mediumincludes a compact disc read-only memory (CD-ROM) as is known in the artfor storing software. The computer-readable medium is accessed by aprocessor suitable for executing instructions configured to be executed.

The following descriptions of various implementations of the presentteachings have been presented for purposes of illustration anddescription. It is not exhaustive and does not limit the presentteachings to the precise form disclosed. Modifications and variationsare possible in light of the above teachings or may be acquired frompracticing of the present teachings. Additionally, the describedimplementation includes software but the present teachings may beimplemented as a combination of hardware and software or in hardwarealone. The present teachings may be implemented with bothobject-oriented and non-object-oriented programming systems.

Scoring Peaks of a Collection of Spectra

As described above, mass spectrometers coupled with separation systemsare used to identify and characterize compounds of interest separatingfrom a sample. The separating sample is ionized and a series of massspectra for the sample are obtained at specified intervals. Inchromatographic systems, the series of spectra collected over time iscalled a chromatogram, for example. For any separation device or system,the series of spectra collected over a number of intervals of aseparation system is referred to herein as a collection of spectra.

Peaks found in the collection of spectra are used to identify orcharacterize a compound of interest in the sample. However, in complexsamples, interfering peaks and approximate or no information regardingthe retention time of the compound of interest can make it difficult toidentify or characterize the compound of interest.

In traditional separation coupled mass spectrometry systems, a fragmention of a known compound of interest is selected for analysis. A massspectrometry scan is then performed at each interval of the separationfor a mass range that includes the fragment ion. The intensity of thefragment ion found in each spectrometry scan is collected over time andanalyzed as a collection of spectra, or an ion chromatogram (XIC), forexample.

For a simple sample mixture, for example, a single peak representing thefragment ion is typically found in the XIC at the expected retentiontime of the known compound. For more complex mixtures, however, two ormore peaks that represent the fragment ion are located at one or moreadditional time intervals in the collection of spectra in addition tothe expected retention time of the compound of interest. In other words,an XIC for the fragment ion can have two or more peaks.

One traditional method of identifying compounds of interest in morecomplex mixtures has been to locate time intervals where two or more ofthe fragment ions of the known compound have peaks. This method is usedin proteomics, for example, when a peptide of a known sequence isquantitated.

In a typical multiple reaction monitoring (MRM) method two or more MRMtransitions are monitored, each corresponding to a different fragment ofthe peptide. If previous discovery data is available, these transitionsare based on the largest fragments that are observed in the data.Otherwise these transitions are based on predicted y-ions, for example.The XIC is analyzed for these two or more MRM transitions. The time atwhich there is a peak for all transitions is used to characterize thecompound of interest.

For complex samples, especially if the expected retention time is notknown accurately, there can be ambiguity in the collection of spectra.For example, there can be more than one retention time or time intervalfor which there is a peak for each of the two or more MRM transitions.

Little additional information is available to address the ambiguityintroduced by complex samples. In traditional separation coupled massspectrometry systems, each mass spectrometry scan for each fragment ionat each time interval is typically performed using a narrow mass windowwidth. As a result, the mass spectrum at a particular time interval foreach fragment ion that is available after data acquisition can providelittle additional insight.

In various embodiments, a separation coupled mass spectrometry system isused that performs mass spectrometry scans at each time interval usingone or more sequential mass window widths in order to span an entiremass range. In other words, spectral information for an entire massrange can be obtained at each time interval in the separation. Recentlydeveloped high-resolution and high-throughput instruments allow a massrange to be accurately scanned within a time interval using multiplescans with adjacent or overlapping mass window widths. Results from themultiple scans can be pieced together to produce a spectrum for theentire mass range at each time interval. The collection of each spectrumat each time interval of the separation is a collection of spectra forthe entire mass range. One exemplary method for using windowed massspectrometry scans to scan an entire mass range is called sequentialwindowed acquisition through libraries (SWATH).

In various embodiments, the spectral information for an entire massrange collected using the windowed acquisition method is used to resolvethe retention time ambiguity in complex mixtures. In other words, when afragment ion is found to have two or more peaks in the collection ofspectra at two or more different time intervals in the separation, amass spectrum of the entire mass range at each of the different timeintervals can be analyzed to determine the actual retention time. Avariety of criteria can be used to analyze the mass spectra of theentire mass range. Based on these criteria each peak and/or timeinterval is scored. A retention time for the known compound isidentified from the peak or peaks with the highest score or combinedscore.

Returning to the proteomics example, a complex sample can have two ormore time intervals where there is a peak for each of the two or moreextracted parent/daughter ion combinations. In other words, a peak grouprepresenting the peptide can be found at two or more time intervals. Invarious embodiments, the mass spectrum for each entire mass range thatwas collected for each of the two or more time intervals is examined.

If the mass accuracy for one or more of the expected masses is poor, forexample, this is an indication that the peak in the collection ofspectra does not correspond to the expected fragment of interest andthis candidate can be eliminated or, in practice, can have its scorereduced. This scoring can be based not just on the two or more initialexpected masses, but on other expected sequence ions for the peptide. Inmany cases, peaks in the collection of spectra correspond to isotopepeaks of other compounds, or to peaks with an incorrect charge state(and hence also unrelated to the compound of interest). For thetraditional MRM method there is no way to detect this situation, butwhen the windowed data acquisition method is used, this situation can bedetected and the corresponding candidate can be ranked more poorly as aresult.

This technique is powerful for peptides since likely sequence ions canbe predicted, but it is also very applicable to small molecules providedthat an initial fragment spectrum is available. In this case one wouldprobably identify the largest observed fragments collection of spectra,but perform scoring using any other significant observed fragments. Oneadditional use of such peak group scoring can be to determine the mostspecific fragment mass or masses, not just for the initial massesidentified in the collection of spectra, but also considering additionalexpected or predicted fragments. This is most useful when “discovery”data is not available so that theoretical peptide y-ions are used orwhen any such discovery data is acquired. If this is done for a sampleknown (or suspected) to contain the peptide of interest, the resultingoptimized fragment masses can be used for subsequent processing (i.e.,XIC calculation) for other samples.

Systems and Methods of Data Processing Separation Coupled MassSpectrometry System

FIG. 2 is a schematic diagram showing a system 200 for scoring peaks ofa known compound of interest from a collection of spectra, in accordancewith various embodiments. System 200 includes separation device 210,mass spectrometer 220, and processor 230. Separation device 210separates one or more compounds from a sample mixture. Separation device210 can include, but is not limited to, an electrophoretic device, achromatographic device, or a mobility device.

Mass spectrometer 220 is a tandem mass spectrometer, for example. Massspectrometer 220 can include one or more physical mass analyzers thatperform two or more mass analyses. A mass analyzer of a tandem massspectrometer can include, but is not limited to, a time-of-flight (TOF),quadrupole, an ion trap, a linear ion trap, an orbitrap, a magneticfour-sector mass analyzer, a hybrid quadrupole time-of-flight (Q-TOF)mass analyzer, or a Fourier transform mass analyzer. Mass spectrometer220 can include separate mass spectrometry stages or steps in space ortime, respectively.

Mass spectrometer 220 performs at each interval of a plurality ofintervals one or more mass spectrometry scans on the separating samplemixture. An interval can include, but is not limited to, a time intervalor an interval of ion mobility. The one or more mass spectrometry scanshave one or more sequential mass window widths in order to span anentire mass range at the interval. As a result, mass spectrometer 220produces a collection of spectra for the entire mass range for theplurality of intervals. This collection of spectra is stored in amemory, for example.

Processor 230 is in communication with tandem mass spectrometer 220.Processor 230 can also be in communication with separation device 210.Processor 230 can be, but is not limited to, a computer, microprocessor,or any device capable of sending and receiving control signals and datato and from tandem mass spectrometer 220 and processing data.

Processor 230 receives the collection of spectra from mass spectrometer220, for example. In various embodiments, processor 230 can receive thecollection of spectra from a file stored in a memory. Processor 230performs the following steps. In step 1, processor selects a fragmention of a known compound. In step 2, processor 230 identifies for thefragment ion one or more peaks at one or more different intervals in thecollection of spectra.

In step 3, processor 230 scores each peak of the one or more peaks.Processor 230 retrieves a mass spectrum of the entire mass range foreach interval of each peak from the collection of spectra. Processor 230compares values of one or more ion characteristics of a mass-to-chargeratio peak in the mass spectrum corresponding to each peak to one ormore known values for the fragment ion. Finally, processor 230 bases thescore of each peak on the results of the comparison.

In various embodiments, the one or more ion characteristics include, butare not limited to, charge state, isotopic state, mass accuracy, or oneor more mass differences associated with a known fragmentation profileof the known compound.

In various embodiments, processor 230 further identifies a separationinterval of the known compound based on scores of the one or more peaks.Processor 230 identifies a separation interval of the known compound asthe interval of a peak of the one or more peaks with the highest score,for example. The separation interval can include, but is not limited to,a retention time in a chromatographic system or an ion mobility at whichthe compound of interest passes through an ion mobility system.

In various embodiments, processor 230 further performs steps 1-3 for oneor more additional fragment ions of the known compound. As a result,processor 230 produces scores for each peak of two or more fragment ionsof the known compound. Processor 230 identifies two or more differentintervals where each fragment ion of the two or more fragment ions has apeak in the collection of spectra. Processor 230 combines scores ofpeaks from the two or more fragment ions at each of the two or moredifferent intervals to produce a combined score for each of the two ormore intervals. Finally, Processor 230 identifies an interval of the twoor more different intervals with the highest score as a separationinterval for the known compound.

In various embodiments, a mass spectrum of the entire mass range fromthe collection of spectra at the separation interval is used forquantitation of the known compound. Alternatively, a mass spectrum ofthe entire mass range from the collection of spectra at the separationinterval is used to locate a modified form of the known compound, forexample.

Mass Spectrometry Method

FIG. 3 is an exemplary flowchart showing a method 300 for scoring peaksof a known compound of interest from a collection of spectra, inaccordance with various embodiments.

In step 310 of method 300, a collection of spectra is obtained for anentire mass range. One or more compounds are separated from a samplemixture using a separation device. One or more mass spectrometry scansare performed on the separating sample mixture at each interval of aplurality of intervals using one or more sequential mass window widthsin order to span the entire mass range. The collection of spectra forthe entire mass range for the plurality of intervals is produced using amass spectrometer. The collection of spectra is obtained directly fromthe mass spectrometer, or indirectly from a file that stores the resultsproduced by the mass spectrometer.

In step 320, a fragment ion of a known compound is selected.

In step 330, for the fragment ion, one or more peaks at one or moredifferent intervals of the plurality of intervals are identified in thecollection of spectra.

In step 340, each peak of the one or more peaks is scored by obtaining amass spectrum of the entire mass range for each interval of each peakfrom the collection of spectra, comparing values of one or more ioncharacteristics of a mass-to-charge ratio peak in the mass spectrumcorresponding to each peak to one or more known values for the fragmention, and basing the score of each peak on the results of the comparison.

Mass Spectrometry Computer Program Product

In various embodiments, a computer program product includes anon-transitory and tangible computer-readable storage medium whosecontents include a program with instructions being executed on aprocessor so as to perform a method for scoring peaks of a knowncompound of interest from a collection of spectra. This method isperformed by a system that includes one or more distinct softwaremodules.

FIG. 4 is a schematic diagram of a system 400 that includes one or moredistinct software modules that perform a method for scoring peaks of aknown compound of interest from a collection of spectra, in accordancewith various embodiments. System 400 includes measurement module 410 andanalysis module 420.

Measurement module 410 obtains a collection of spectra for an entiremass range. One or more compounds are separated from a sample mixtureusing a separation device. One or more mass spectrometry scans areperformed on the separating sample mixture at each interval of aplurality of intervals using one or more sequential mass window widthsin order to span the entire mass range. The collection of spectra forthe entire mass range is produced for the plurality of intervals using amass spectrometer.

Analysis module 420 selects a fragment ion of a known compound. Analysismodule 420 identifies for the fragment ion one or more peaks at one ormore different intervals of the plurality of intervals in the collectionof spectra. Finally, analysis module 420 scores each peak of the one ormore peaks. A mass spectrum of the entire mass range for each intervalof each peak is obtained from the collection of spectra. Values of oneor more ion characteristics of a mass-to-charge ratio peak in the massspectrum corresponding to each peak are compared to one or more knownvalues for the fragment ion. The score of each peak is based on theresults of the comparison.

While the present teachings are described in conjunction with variousembodiments, it is not intended that the present teachings be limited tosuch embodiments. On the contrary, the present teachings encompassvarious alternatives, modifications, and equivalents, as will beappreciated by those of skill in the art.

Further, in describing various embodiments, the specification may havepresented a method and/or process as a particular sequence of steps.However, to the extent that the method or process does not rely on theparticular order of steps set forth herein, the method or process shouldnot be limited to the particular sequence of steps described. As one ofordinary skill in the art would appreciate, other sequences of steps maybe possible. Therefore, the particular order of the steps set forth inthe specification should not be construed as limitations on the claims.In addition, the claims directed to the method and/or process should notbe limited to the performance of their steps in the order written, andone skilled in the art can readily appreciate that the sequences may bevaried and still remain within the spirit and scope of the variousembodiments.

What is claimed is:
 1. A system for scoring peaks of a known compound ofinterest from a collection of spectra, comprising: a separation devicethat separates one or more compounds from a sample mixture; a massspectrometer that performs at each interval of a plurality of intervalsone or more mass spectrometry scans on the separating sample mixtureusing one or more sequential mass window widths in order to span anentire mass range producing a collection of spectra for the entire massrange for the plurality of intervals; and a processor that (a) selects afragment ion of a known compound, (b) identifies for the fragment ionone or more peaks at one or more different intervals of the plurality ofintervals in the collection of spectra, and (c) scores each peak of theone or more peaks by obtaining a mass spectrum of the entire mass rangefor each interval of the each peak from the collection of spectra,comparing values of one or more ion characteristics of a mass-to-chargeratio peak in the mass spectrum corresponding to the each peak to one ormore known values for the fragment ion, and basing the score of the eachpeak on the results of the comparison.
 2. The system of claim 1, whereinthe plurality of intervals comprises a plurality of intervals.
 3. Thesystem of claim 1, wherein the plurality of intervals comprises aplurality of ion mobilities.
 4. The system of claim 1, wherein the oneor more ion characteristics comprise charge state.
 5. The system ofclaim 1, wherein the one or more ion characteristics comprise isotopicstate.
 6. The system of claim 1, wherein the one or more ioncharacteristics comprise mass accuracy.
 7. The system of claim 1,wherein the one or more ion characteristics comprise one or more massdifferences associated with a known fragmentation profile of the knowncompound.
 8. The system of claim 1, wherein the processor furtheridentifies a separation interval of the known compound based on scoresof the one or more peaks.
 9. The system of claim 8, wherein theseparation interval comprises a retention time.
 10. The system of claim8, wherein the separation interval comprises an ion mobility.
 11. Thesystem of claim 8, wherein the processor identifies a separationinterval of the known compound as the interval of a peak of the one ormore peaks with the highest score.
 12. The system of claim 1, whereinthe processor further performs steps (a)-(c) for one or more additionalfragment ions of the known compound producing scores for peaks of two ormore fragment ions of the known compound, identifies two or moredifferent intervals of the plurality of intervals where each fragmention of the two or more fragment ions has a peak in the collection ofspectra, combines scores of peaks from the two or more fragment ions ateach of the two or more different intervals to produce a combined scorefor each of the two or more intervals, and identifies an interval of thetwo or more different intervals with the highest score as a separationinterval for the known compound.
 13. The system of claim 12, wherein amass spectrum of the entire mass range from the collection of spectra atthe separation interval is used for quantitation of the known compound.14. The system of claim 12, wherein a mass spectrum of the entire massrange from the collection of spectra at the separation interval is usedto locate a modified form of the known compound.
 15. A method forscoring peaks of a known compound of interest from a collection ofspectra, comprising: obtaining a collection of spectra for an entiremass range, wherein one or more compounds are separated from a samplemixture using a separation device and wherein one or more massspectrometry scans are performed on the separating sample mixture ateach interval of a plurality of intervals using one or more sequentialmass window widths in order to span the entire mass range producing thecollection of spectra for the entire mass range for the plurality ofintervals using a mass spectrometer; selecting a fragment ion of a knowncompound; identifying for the fragment ion one or more peaks at one ormore different intervals of the plurality of intervals in the collectionof spectra; and scoring each peak of the one or more peaks by obtaininga mass spectrum of the entire mass range for each interval of the eachpeak from the collection of spectra, comparing values of one or more ioncharacteristics of a mass-to-charge ratio peak in the mass spectrumcorresponding to the each peak to one or more known values for thefragment ion, and basing the score of the each peak on the results ofthe comparison.
 16. A computer program product, comprising anon-transitory and tangible computer-readable storage medium whosecontents include a program with instructions being executed on aprocessor so as to perform a method for scoring peaks of a knowncompound of interest from a collection of spectra, the methodcomprising: providing a system, wherein the system comprises one or moredistinct software modules, and wherein the distinct software modulescomprise a measurement module and an analysis module; obtaining acollection of spectra for an entire mass range using the measurementmodule, wherein one or more compounds are separated from a samplemixture using a separation device and wherein one or more massspectrometry scans are performed on the separating sample mixture ateach interval of a plurality of intervals using one or more sequentialmass window widths in order to span the entire mass range producing thecollection of spectra for the entire mass range for the plurality ofintervals using a mass spectrometer; selecting a fragment ion of a knowncompound using the analysis module; identifying for the fragment ion oneor more peaks at one or more different intervals of the plurality ofintervals in the collection of spectra using the analysis module; andscoring each peak of the one or more peaks using the analysis module byobtaining a mass spectrum of the entire mass range for each interval ofthe each peak from the collection of spectra, comparing values of one ormore ion characteristics of a mass-to-charge ratio peak in the massspectrum corresponding to the each peak to one or more known values forthe fragment ion, and basing the score of the each peak on the resultsof the comparison.